High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration.

CRISPR Cas9 system is becoming an emerging genome-editing platform and has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. In this study, we developed a novel replicative and integrative CRISPR Cas9 genome-editing platform for large DNA construct in vivo assembly, replication, and high-copy genome integration in Saccharomyces cerevisiae. It harnessed advantages of autonomous replicative sequence in S. cerevisiae, in vivo DNA assembly, CRISPR Cas9, and delta integration. Enhanced green fluorescent protein was used as a marker to confirm large DNA construct in vivo assembly and genome integration. Based on this platform, an efficient 2,3- BDO producing yeast strain was rapidly constructed with up to 25-copy genome integration of 2,3-BDO biosynthesis pathway. Further strain engineering was conducted by multiplex disruption of ADH1, PDC1, PDC5 and MTH1 using a 2µ-based replicative CRISPR Cas9 plasmid containing donor DNAs. As a result, the 2,3-BDO titer was improved by 3.9 folds compared to that obtained by the initially engineered yeast and 50.5 g/L 2,3-BDO was produced by the final engineered yeast strain 36aS5-CFBDO in fed-batch fermentation without strain evolution and process optimization. This study demonstrated that the new replicative and integrative CRISPR Cas9 genome-editing platform was promising in generating an efficient 2,3-BDO-producing S. cerevisiae strain.

Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain.

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.47, 0.38 and 1.62 g/g DW/h, with an ethanol titre of 41.07 g/L and yield of 0.42 g/g. Industrial wheat straw hydrolysate fermentation resulted in maximal specific rates of glucose and xylose consumption, and ethanol production of 2.61, 0.54 and 1.38 g/g DW/h, respectively, with an ethanol titre of 54.11 g/L and yield of 0.44 g/g. These are among the best for wheat straw hydrolysate fermentation through separate hydrolysis and cofermentation.

Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

BACKGROUND: Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation. RESULTS: Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw(-1) h(-1), and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw(-1) h(-1), much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene. CONCLUSIONS: This study demonstrated that XIs clustered in the mammal gut Bacteroidetes group were able to be expressed functionally in S. cerevisiae and background strain anaerobic adaptive evolution in xylose medium is essential for the screening of functional XIs. The methods outlined in this paper are instructive for the identification of novel XIs that are functional in S. cerevisiae.